The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome from one cell generation to the next, DNA must be duplicated in a highly controlled and accurate manner. Interruption of these controls may translate to more rapid progression of neoplastic transformation. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention of proliferation of pathogenic eukaryotes. In recent studies the DNA sequences and initiator protein that cooperate to determine the location of origins of DNA replication in Saccharomyces cerevisiae have been identified. The initiator protein is a multi-subunit, sequence-specific DNA binding protein. It is hypothesized to interact with other proteins that are required for the initiation of DNA replication. Proposed research in this application will investigate how DNA replication occurs in an ORC dependent manner and will study how initiation of DNA replication is temporally controlled during progression through the cell division cycle. In specific alm 1, proteins that interact with the DNA sequences that constitute the origin of DNA replication will be identified. In specific aim 2, proteins that interact with ORC will be identified by biochemical and genetic methods. From studies on virus DNA replication, the DNA polymerase alpha-primase complex has been demonstrated to function in the initiation of DNA replication. In specific aim 3, proteins that interact with this essential polymerase will be identified. Specific aim 4 has a goal of establishing a soluble cell-free system for replication of chromosomal DNA. Finally, specific aim 5 deals with the role of the identified initiation proteins as targets of the known regulatory proteins that control progression through the cell division cycle.